Assay system

ABSTRACT

An assay apparatus comprising: i) an assay cartridge ( 52, 53 ) comprising at least one well ( 57 - 62 ) and a pipette ( 50 ) positionable in at least one said well; ii) a holder arranged to receive said cartridge; iii) drive means operable to position said pipette in selected wells of said cartridge; iv) a gas pressure applicator couplable to said pipette whereby to cause liquid flow through said membrane; and v) a radiation detector operable to detect radiation from a well of said cartridge or from said pipette.

[0001] This invention relates to improvements in and relating to assaysystems, especially diagnostic assay systems, in particular systemsusable at the point-of-care, e.g. at the physician's place of work or atthe patient's bedside.

[0002] Many diagnostic assays are currently available, e.g. assays forpregnancy, blood sugar, homocysteine, carbohydrate-deficienttransferrin, blood-clotting, blood cholesterol, etc. Some such assaysare performable by the patient, and some by the patient's physician, butmany, especially those which provide a quantitative result, mustcurrently be performed in a laboratory remote from both patient andphysician and so result in significant delays between sampling andtesting and generally require the patient to make a further visit to thephysician to learn the assay's results. This is not only inconvenient tothe patient but also increases the costs to the patient or theorganization paying for the patient's health care.

[0003] There is thus an ongoing need for assay systems, especially onesproviding quantitative results, operable by the physician or thephysician's colleagues at the point of patient care.

[0004] Quantitative assay systems often require highly accurate volumemeasuring devices, several reagents, and assay-specific result-readingdetectors, and it is impractical to provide dedicated assay apparatusfor a wide range of different assay systems at the point of care, bothfor reasons of space and expense.

[0005] We have therefore developed an assay apparatus which, inpreferred embodiments, is capable of use at the point of care, iscapable of performing a range of different assays, is capable ofyielding quantitative assay results, and is relatively inexpensive.

[0006] Viewed from one aspect the invention provides an assay apparatus,preferably a diagnostic assay apparatus, comprising:

[0007] i) an assay cartridge comprising at least two wells and a pipettepositionable in at least two of said wells, said pipette having aproximal end and a distal end, said distal end being closed by a liquidpermeable membrane;

[0008] ii) a holder arranged to receive said cartridge;

[0009] iii) drive means operable to position said pipette in selectedwells of said cartridge;

[0010] iv) a gas pressure applicator couplable to said pipette wherebyto cause liquid flow through said membrane;

[0011] v) a radiation detector operable to detect radiation from a wellof said cartridge or from said pipette; and, optionally but preferably,

[0012] vi) an electromagnetic radiation source.

[0013] Viewed from a further aspect the invention provides an assaycartridge comprising at least two wells and a pipette positionable in atleast two of said wells, said pipette having a proximal end and a distalend, said distal end being closed by a liquid permeable membrane.

[0014] A pipette is a tube with an aperture at one end (the distal end)into which a liquid may flow on application of a reduced pressure to theother end (the proximal end). In the apparatus referred to in thepreceding paragraphs the distal end of the pipette is tipped with(closed by) a liquid permeable membrane. The proximal end of thispipette may be open or closed but if closed then clearly this must be bysome means which allows the pressure application necessary for thepipette to function as a pipette. In one embodiment described below, theproximal end of the membrane-tipped pipette is sealed with a pierceableself-sealing membrane (e.g. a rubber gasket) and pressure may be appliedthrough a hollow needle inserted through the membrane. Alternatively theproximal end may be closed by a removeable cap or stopper which isremoved to allow pressure application, or by a frangible seal which isbroken to allow pressure application.

[0015] Viewed from a still further aspect the invention provides anassay device comprising a) a cartridge holder capable of receiving anassay cartridge according to the invention; b) drive means operable toposition the pipette of a said cartridge in selected wells of saidcartridge; c) a gas pressure applicator couplable to the pipette of asaid cartridge whereby to cause liquid flow therethrough; d) a radiationdetector operable to detect radiation from a well of a said cartridge orfrom the pipette thereof; and, optionally but preferably, e) anelectromagnetic radiation source.

[0016] Thus the combination of the device and the cartridge of theinvention provides an assay apparatus according to the invention.

[0017] The assay cartridge preferably is provided to the user pre-filledwith the reagents required for the particular assay or assays to beperformed using that cartridge. Where two or more reagents are requiredand these should not be mixed before the assay is performed, these maybe pre-filled into different wells in the cartridge. Generally suchreagents will be prefilled into the wells in measured quantities. Suchreagents may for example be liquids, powders, beads, coatings on thewell walls, coatings on beads, or materials impregnated into orimmobilized on the membrane of the pipette. Where the reagents areliquid or where they are susceptible to degradation on exposure to airor moisture, the cartridge may be sealed to prevent liquid loss or airor moisture access to the susceptible reagent. Such sealing isconveniently achieved by forming the cartridge with a well-containingbase and a well-covering cap, and if necessary placing a fluidimpermeable seal, e.g. an O-ring, between the well-openings in the baseand the well-covering cap, and if desired by placing a removable-seal,e.g. an adhesive sealing strip, about the external junction between capand base. In another more preferred embodiment, one or more of the wellsmay be foil sealed before use: in this embodiment the well covering capis preferably equipped with foil seal cutters for cutting thewell-covering foil seals to permit the pipette to be inserted into thesewells. Alternatively, the cap may be provided with resilient material atpositions corresponding to the tops of the wells (or just the liquidcontaining wells) such that when cap and base are urged together, aliquid-tight seal is formed at the well tops. Such material may forexample be a layer coated onto the cap or discs or gaskets attached(e.g. welded or adhered) to the cap. In one embodiment, the lowersurface of the cap is provided with resilient projections capable offunctioning as stoppers for the wells. In this way, the stoppers serveto keep cap and base together before use of the cartridge in an assayand after assay performance base and cap can be sealed for disposalsimply by urging the two together causing the stoppers again to seal thewells. This is particularly advantageous when the wells following assayperformance contain toxic or potentially infectious materials. Such capscan, if desired, be removed before use; however, in a preferredembodiment, the cap will serve to hold the pipette and possibly also toprovide attachment means for the pressure applicator. In such anembodiment the drive means may serve to move base relative to cap so asto position the pipette in the desired wells in the different stages ofthe assay.

[0018] In general, and particularly where the cartridge cap is providedwith resilient stoppers for the wells in the cartridge base, theapparatus and device of the invention preferably comprise means forseparating the cap from the base so that the cartridge may be loadedinto the device still sealed. In one embodiment, such separating meanscomprises a wedge which is moved past the loaded cartridge and engageswith projections, e.g. flanges, on cap and base to force the two apart.Desirably this separating means is automatically brought into operationfollowing cartridge loading, e.g. in response to the shutting of the lidto the chamber containing the loaded cartridge or on transport of thecartridge into the chamber for example using a conveyor which cansimilarly remove the cartridge from the chamber following assayperformance.

[0019] For different assays, e.g. for different analytes, differentassay cartridges may be provided; however, cartridges may be designedfor performance of two or more different assays. In this latter case, itwill frequently be desirable for the cartridge to contain two or moremembrane-capped pipettes, i.e. so that a different pipette can be usedfor each of the assays.

[0020] The wells in the cartridge may be provided in any desiredpattern, e.g. as a two dimensional array (e.g. as in conventionalmulti-well plates), as a linear array, or as a circular array. The useof circular and especially linear arrays is particularly preferred asthe mechanism required for moving the cartridge between preset positionsis simplified, i.e. the drive means may then operate to move thecartridge along a linear path or to rotate the cartridge.

[0021] The use of a linear array of wells is especially preferred,particularly an array comprising, in sequence: a material handling well(optionally before use storing a capillary-tipped pipette removablymounted on the cartridge cap or adapted to receive during use acapillary-tipped pipette mountable on the cartridge cap); a well whichbefore use stores the membrane-tipped pipette or a further capillarytipped pipette mounted on the cartridge cap; and one or a series of twoor more (e.g. up to six) wells for assay performance and assay resultreading—these wells may contain reagents and before use such reagentcontaining wells may be foil sealed and one of these wells may be openended or open-sided to facilitate result reading. In such anarrangement, the cap and base may desirably be separated before assayperformance begins and re-engaged only when assay performance iscompleted. Thus result reading in this arrangement takes place while capand base are disengaged from each other. In this arrangement, the capand base are preferably latched together, e.g. by a snap-lock latch. Thematerial handling well may for example contain dry reagent for mixingduring assay performance, a filter for sample separation (e.g. to removeerythrocytes from a blood sample), or a further pipette capable ofmating engagement with a cap-mounted pipette (e.g. a capillary-tippedpipette).

[0022] While the cartridge must contain at least two wells, one or morepositions in the well array of a multi-well cartridge may be open-endedor open-sided such that detection of radiation from the pipette whenlocated in such positions is facilitated. If radiation from a pipette ina well is to be detected, then at least a portion of the well wall mustbe transparent to the type of radiation to be detected.

[0023] The wells in the cartridge may remain stationary during theassay; however, as it may be desirable to use the detector to monitorthe progress of the assay, it is generally preferable that the drivemeans is operable to move the cartridge between two or more pre-setpositions so that the detector can detect radiation from differentcartridge wells. Alternatively but less preferably, the detector itselfmay be movable between pre-set positions or movable mirrors may beprovided so as to permit the light path from cartridge to detector to bevaried to achieve the same effect.

[0024] Thus in a preferred embodiment the drive means will operateduring the assay to lift the cartridge cap and pipette away from thewell-containing base (or more preferably to drop the base away from thecap), to move the base relative to the cap (preferably by moving thebase, e.g. linearly or by rotation) to bring the pipette into registrywith the desired well, and to move cap and base together to place thepipette into the desired well, and so on until the assay is complete.

[0025] In some assays it may be desirable to tilt the wells duringliquid transfer or to agitate liquid in a well and accordingly it isdesirable that the drive means also be operable to tilt or agitate (e.g.rock or shake) at least the well-containing portion of the cartridge.

[0026] The drive means may be manually operable, e.g. a mechanical driveor a motor driven drive activated at each stage by the operator; howeverit will preferably be a motor drive activated to perform the requiredactions by an external or more preferably internal computer whichoperates the assay apparatus.

[0027] The wells in the cartridge may be of any desired shape or volume;however preferably they will be straight-sided cylindrical or lesspreferably tapered cylindrical. The cross-section of such cylindricalwells may be of any desired shape, e.g. circular, oval, polygonal (e.g.rectangular), semicircular, etc. The well bases may be flat or curved;however for wells which are to be monitored from below during or at theend of the assay, the well base will preferably be flat. In aparticularly preferred embodiment, the well base is flat and sloping,i.e. non-horizontal. The wells may be within a solid base oralternatively, and less preferably, the wells may be connected in astrip, plate, disc, daisy-wheel, etc. format. The well walls, forexample the solid well-containing base, will preferably be of plastic,especially light-transparent plastic, e.g. acrylic, vinylic, styrenic orolefinic plastic. The choice of the particular plastic will howeverdepend, as is conventional, on the nature of the reagents used. It hasbeen found particularly preferable to use plastics with good opticalproperties and low gas and/or liquid permeability. To this end,copolymers of alpha-olefins (e.g. ethylene and propylene, especiallyethylene) and cyclic olefins (e.g. norbornene) are especially preferred,e.g. the product sold under the trade name Topas® 8007 by Ticona GmbH ofFrankfurt, Germany (Topas® 8007 is an ethylene/norbornene copolymer).Desirably such copolymers have a light transmission (measured accordingto ASTM D1003 for a 2 mm wall thickness) of at least 80%, mostpreferably at least 90%; and a water vapour permeability (at 23° C. and85% RH, measured according to DIN 53122 on a 80×80×1 mm sample) of lessthan 0.2 g.mm.m⁻²d⁻¹, more preferably less than 0.05 g.mm.m⁻²d⁻¹.

[0028] Typically, the wells will have internal diameters of 3 to 20 mm,especially 5 to 15 mm, and volumes of 0.1 to 5 mL, especially 0.5 to 1.5mL.

[0029] The membrane-tipped pipette in the cartridge of the invention ispreferably cylindrical and the membrane is preferably at or morepreferably covering one end. The other, open, end is preferably shapedfor substantially gas-tight attachment to a pressure applicator. Thepipette may be of any appropriate material; however transparent plasticor glass is preferred. The membrane may be attached to the pipette inany appropriate fashion, e.g. by welding (e.g. ultrasonic or thermalwelding), adhesive, fusion of a granular membrane precursor, etc.

[0030] The membrane itself may be of any appropriate material, e.g.plastics (e.g. nylon, polysulphones, etc.), glass (e.g. glass fibre),metal, etc. However cellulosic-membranes (e.g. reinforcednitrocellulose) are especially preferred as it is relativelystraightforward to immobilize antibodies or other assay reagents on suchmaterials.

[0031] In various embodiments of the invention, the membrane ispreferably planar and perpendicular to the pipette axis; such membranesare particularly effective for removal of liquid from a horizontal flat-or concave-bottomed well.

[0032] The membrane however may alternatively and more preferably beplanar but angled relative to the axis of the pipette, e.g. up to 85°off perpendicular to the axis, preferably 10 to 80° off perpendicular,more preferably 50 to 70° off perpendicular, especially about 60° offperpendicular. Where the pipette and one or more of the wells isrectangular (e.g. square) in cross-section, it is preferred that themembrane be angled and that the base of one or more such wells likewisebe angled so as to be substantially parallel to the membrane when thepipette is in that well.

[0033] The use of a sloping membrane is especially advantageous as for agiven pipette cross-sectional area, the surface area of the membrane isincreased as it is angled progressively further from the horizontal, sogiving a larger surface area to be read or monitored during the assay.Most surprisingly, not only do sloping membranes allow essentially allof the contents of a correspondingly shaped well to be taken up throughthe membrane but also the uptake is uniform across the membrane (i.e. ifa coloured analyte becomes trapped on the membrane the membrane becomesuniformly coloured). A further advantage is that the membrane may beviewed from the side avoiding any risk of droplets of sample, reagent,etc., falling onto the apparatus optics. A still further advantage isthat the membrane may readily be illuminated without causing highincidence of the illuminant light being reflected into the lightdetector. Another advantage is that, even with a coloured sample (e.g.blood), it is possible to monitor the membrane surface through the wellside wall and thus to terminate any reaction step when the desiredchange in membrane surface has occurred as the membrane-to-well wallspacing can be less than that for a horizontal membrane in aliquid-containing well. A yet still further advantage is that theformation of bubbles between the membrane and the facing well wall isreduced relative to the case for horizontal membranes so reducing theneed to tilt or shake the cartridge base.

[0034] The use of angled membrane tipped pipettes is thought to be noveland thus viewed from a further aspect the invention provides a pipettethe distal end whereof is cylindrical and tipped by a porous membranethe outer surface whereof is angled away from the plane perpendicular tothe cylindrical axis of said distal end, said pipette preferably formingpart of a diagnostic assay cartridge.

[0035] The use of a rectangular cross-section for a well is especiallypreferred as it reduces the incidence of liquid reagents being trappedat the upper end of wells by capillary effects following inversion ofthe assay cartridges during transport or storage. The corners where wellside walls meet should therefore desirably be as sharp as possible atthe upper ends of the wells, e.g. having a radius of curvature of 0.5 mmor less, e.g. 0.1 mm or less. However, to prevent liquids in the base ofthe wells creeping up the corners of the well, it is desirable that atthe lower end of the wells the corners should be chamfered or morerounded, e.g. having a radius of curvature of at least 0.5 mm,preferably at least 0.8.mm.

[0036] Where a well is to be used for assay reading, e.g. where theabsorption of light passing through a liquid in the well is to bemeasured, it is also particularly preferred to use a rectangularcross-section well with an angled base. In this way, by appropriatemasking of the section of the well visible to the detector, one maychoose to measure light transmitted through the full width of the wellor through a narrower width at the base of the well (i.e. between a sidewall and the sloping base). Thus the light path length through the wellmay be increased or decreased by moving the visible section up or down.In this way, for example, where the optical density of the well-contentsis high, a shorter path length may be chosen.

[0037] Moreover, by measuring light transmission intensity at two ormore path lengths (e.g. within and above the tapered base portion of thewell), the contribution of the well walls to the detected signal can bedetermined and corrected for.

[0038] Where scattered light is to be detected (e.g. where the samplebeing read contains particles or agglomerates or is fluorescent orphosphorescent), it will again be desirable to use rectangularcross-section wells with the incident light being directed perpendicularto one pair of well walls and with the scattered light being detected bya detector (e.g. digital camera) directed at one of the other walls.Where the cartridge contains a linear array of wells, the reading wellfor light scattering measurements is preferably at one end of the array.

[0039] This use of angled wall wells is also novel and forms furtheraspects of the invention.

[0040] Viewed from a further aspect the invention thus provides an assayapparatus comprising:

[0041] i) an assay cartridge comprising at least one well and a pipettepositionable in at least one said well, at least one said well havingtwo parallel planar side walls joined by a base wall comprising at leastone planar face the normal to the surface whereof is coplanar to andnon-perpendicular to normals to the parallel planar surfaces of saidside walls;

[0042] ii) a holder arranged to receive said cartridge;

[0043] iii) drive means operable to position said pipette in selectedwells of said cartridge;

[0044] iv) a gas pressure applicator couplable to said pipette wherebyto cause liquid flow through said membrane; and

[0045] v) a radiation detector operable to detect radiation from a wellof said cartridge or from said pipette.

[0046] In this aspect the base is preferably planar, angled to thehorizontal as described above, and the well is preferably rectangular incross-section. The cartridge moreover preferably contains at least onecapillary-tipped pipette and/or membrane-tipped pipette, again asdescribed herein.

[0047] Viewed from a still further aspect the invention provides anassay cartridge comprising at least one well and a pipette positionablein at least one said well, at least one said well having two parallelplanar side walls joined by a base wall comprising at least one planarface the normal to the surface whereof is coplanar to andnon-perpendicular to normals to the parallel planar surfaces of saidside walls.

[0048] In addition to a membrane-tipped pipette, the cartridges of theinvention may contain one or more further pipettes, again preferablycarried by the cartridge cap, for example for measuring out an accuratevolume of reagent or sample or for mixing reagents and samples. In onepreferred embodiment the cartridge contains a capillary-tipped pipettewhich draws up a desired amount of fluid from a sample by virtue of itscapillary action. Particularly desirably this comprises a capillaryopening into a chamber of wider internal diameter such that capillaryaction causes only the capillary tip to fill. With the tipwithdrawn-from the surrounding liquid, the contents of the tip can thenbe ejected into a cartridge-well under pressure or sucked up furtherinto the pipette beyond the capillary tip and chamber.

[0049] In another aspect of the invention, the cartridge may comprise acapillary-tipped pipette in place of the membrane-tipped pipette. Aswill be discussed further below, such a cartridge may for example beused in a clotting time assay.

[0050] The external diameter of the membrane-tipped pipette ispreferably at least 0.8 mm, e.g. 1 to 5 mm, especially 1.5 to 2.5 mm,less than the internal diameter of the wells so as to facilitate gasflow between well wall and pipette during liquid transfer across thepipette membrane and to ensure substantially complete uptake of-liquidfrom the wells. The gap also allows the well to contain liquid (e.g. 200μL) and the membrane-tipped pipette before uptake of liquid into thepipette.

[0051] While the pipette and the wells may have the same form ofcross-sectional shape (i.e. circular, square, etc.), it may occasionallybe preferred that the shapes differ slightly, e.g. one being circularand the other elliptical, as this reduces the risk of themembrane-tipped pipette being held by suction to the bottom of a well.This problem may similarly be addressed by making the pipette tip or thewell base slightly irregular, e.g. with indentations or projections.

[0052] In a particularly preferred embodiment, the cartridge comprises:a base containing a plurality, e.g. 2 to 8 or 10, of wells, at least twoand preferably at least 3 of which are free of liquid reagents and atleast one of which contains a liquid reagent; and a cap carrying themembrane-tipped pipette such that it is disposed with the membrane endin one of the empty wells and with the open end accessible on the outersurface of the cover, and having a sample application aperture throughthe cover to communicate with another of the liquid-free wells.Desirably removable seals are provided to cover the open ends of thepipette and the sample application aperture. Unless the cap carrieswell-sealing stoppers or the wells are sealed as described above, afurther removable seal will preferably be provided to surround theexternal junction of cap and base and O-ring or other seals will beprovided around at least the liquid containing wells between cap andbase. In either of these ways the interior of the cartridge is isolatedfrom air and moisture before use. The base and cap preferably haveindentations or projections for engagement with the cartridge holder anddrive means, for ensuring correct registry between cap and base duringassay performance, and if the cap carries-well-sealing stoppers, forengagement with a separator such as-described above which operates toseparate cap and base to allow the assay to proceed.

[0053] The base and cap are preferably such that the membrane-tippedpipette can be placed within a “reading well” or in a well-free positionat which radiation from the pipette is accessible to the detector. Sucha “reading well” may for example have a light-transparent flat base orflat side well section through which light may pass to the detector. Inthe case where reading is at a well-free position, this may for examplebe an open-ended aperture through the base or a portion of the basewhere its side wall is removed or recessed such that light from thepipette may reach the detector without passing through the material fromwhich the base is formed.

[0054] The use of a “reading well” is preferred since the possibility ofreagents or sample dripping into the body of the assay apparatus isreduced. Where an angled membrane is to be read, the use of a separatereading well may be avoided as simply lifting the membrane out of theliquid in a well or sucking the liquid through the membrane into thepipette leaves the membrane surface exposed for reading.

[0055] In one embodiment, the base may be formed to provide a mirrorsurface (e.g. a plastic prism surface) under the bottom of the readingwell which reflects light from the bottom of the reading well, e.g. fromthe vertical to the horizontal. In this way, the detector need not bepositioned below the cartridge and problems of dust or liquid fallingonto the detector may be avoided. As in a Fresnel lens, a prism maysimilarly be produced as an integral combination of parallel individualprism elements. This prism structure is referred to herein as a “Fresnelprism”, and such prisms and their uses, e.g. as light path modifiers inoptical apparatus, for example assay devices, form further aspects ofthe present invention. Image distortion, due to surface distortion oftenseen in plastic mouldings with a thickness of more than few millimetres,is reduced or avoided by use of a plastic Fresnel prism rather than aconventional plastic prism having the same light incidence surface area.Thus the use of a Fresnel prism formed in the cartridge base to achievelight reflection is especially preferred in the devices of theinvention. A typical “Fresnel prism”, is a structure of transparentmaterial stepped on one side and flat on the other—light incidentnormally on the horizontal part of a step is internally reflected by theflat surface and leaves normally through the vertical part of a step. Ineffect therefore it functions as a mirror. With an angled membranehowever such a Fresnel prism will not generally be needed.

[0056] In the cartridges of the invention, the proximal or “open” end ofat least one pipette is preferably sealed with a resilient self-sealingmembrane, e.g. a rubber membrane, which may be pierced by a hollowneedle to allow gas pressure application. In this embodiment, a wastereservoir is preferably disposed in the pipette between the pipette tipand the resilient membrane. With this embodiment, liquid in thecartridge may be drawn up into the waste reservoir during or at the endof assay performance so that the used cartridge may be removed anddisposed of without waste leakage occurring.

[0057] The gas pressure applicator in the apparatus of the invention mayfor example comprise a pump, and a conduit from the pump to a cartridgeattachment, and optionally at least one reservoir and a two or moreposition valve. Inclusion of a reservoir, e.g. of one or more litrecapacity, and preferably at least two reservoirs, allows pressures aboveand/or below ambient to be applied to the pipette for short durationswith negligible time variation of the pressure applied due to theability to isolate the pipette from the pump and due to the relativelysmall pressure change within the reservoir during the pressureapplication period (as a result of the relatively large size of thereservoir). Between pressure applications, the pump can be used to bringthe reservoir pressure back to the desired level. Since it may bedesirable to vent the pipette to atmospheric and/or to provide pressuresabove and below ambient to the pipette, it is desirable to place amulti-position valve in the conduit upstream of the pipette to allowsuch different pressure applications. The valve, which should desirablyalso include a closed position allowing no gas flow to or from thepipette, is preferably computer operated. The use of pressure reservoirsas described above however results in a relatively large spacerequirement for the apparatus and device of the invention. Since thedevice is preferably portable, it is preferred instead to use apiston-based pump (e.g. a syringe) coupled via a conduit (preferably ofminimal volume) to a cartridge attachment. Indeed it is especiallypreferred to have an array of coupled piston-pumps, each connected to aseparate cartridge attachment so that, when the cartridge is in place,operation of a pump motor causes all of the pumps to operate. In thisembodiment, the cartridge is preferably provided with blank or activemeans for engaging each of these attachments, the blank engaging meanssimply allowing the respective piston pump to vent. In certainembodiments, for example in clotting time measurements or where ananalyte is required to bind to a ligand immobilized on the pipettemembrane, it may be desirable to speed up or slow down passage of liquidunder the influence of the pressure applicator; in these circumstancesthis may for example be achieved by speeding up or slowing down thespeed of the pistons in the piston-pumps.

[0058] The pressure applicator is preferably coupled directly to theopen end of the pipette; however alternatively and much less preferablyit may be coupled directly to a well in the cartridge with the open endof the pipette open to ambient pressure.

[0059] In one particular embodiment, a (preferably moveable) pressureapplicator attachment is provided for each well or well-free-readingposition-of the cartridge and the cartridge is provided with blank oractive means for engaging each of these attachments. In this way it maybe possible to avoid the need for careful orientation of the cartridgeduring placement in the holder—the cartridge could be placed in any oneof the pre-set permitted orientations and the lid of the apparatusclosed to bring the attachments automatically into engagement with theblank and active engagement means on the cartridge. Cartridgeidentification (as discussed further below) by the apparatus would thenallow the cartridge to be moved automatically into the correctorientation for commencement of the assay. This however is onlyespecially desirable if it is important to reduce the time required forcartridge placement or if the cartridge is designed for use in multipleassays (i.e. has multiple pipettes).

[0060] The detector in the apparatus of the invention may be anyappropriate radiation detector, e.g. a radioactive emission detector oran electromagnetic radiation detector. Alternatively the apparatus maycontain two or more detectors capable of detecting different types ofradiation. However, for point of care use, it is preferred that thedetector be an electromagnetic radiation detector and more specificallya detector capable of detecting light in at least part of the UV to IRrange, particularly the near UV to near IR range and more especially thevisible range. (The term light is used here to mean electromagneticradiation in the UV to IR range.) For this purpose it is especiallypreferred to use a digital camera as the detector.

[0061] The use of a digital camera as the detector is especiallypreferred since it can function not only as a light detector but as animage structure analyser. Thus, for example, irregularities in the imageof a membrane on a pipette may be detected and corrected for.

[0062] Between detector and cartridge it may be desirable to place,movably or fixedly, items which serve either to select the radiationenergy allowed to pass to the detector (e.g. filters, prisms, etc.) orto reduce stray radiation impact on the detector (e.g. apertures andlight traps).

[0063] Stray radiation reducing items are especially important where theradiation to be detected is weak (e.g. resulting from chemoluminescenceor fluorescence) or stimulated or results from transmission orreflection of radiation measurable by the detector. In suchcircumstances, light barriers or collimators may also be providedelsewhere in the apparatus or within the cartridge.

[0064] In general, the apparatus of the invention will be provided withelectromagnetic radiation sources (e.g. sources of visible light or nearIR to near UV), disposed to cause radiation emitted, reflected ortransmitted by the desired cartridge wells or pipette to pass to thedetector. As a result it is also preferred that cartridge, cartridgeholder and detector be disposed in a light proof chamber in theapparatus and that the apparatus be provided with a closable access portfor cartridge placement, e.g. a lid.

[0065] It is especially preferred that a light source be provided which,when the cartridge is in place, has a well between it and the detector,e.g. so that light transmittance in the well may be determined. For thispurpose, the cartridge may be provided with an aperture into which sucha light source may be inserted on cartridge loading, preferably anaxially positioned aperture where the wells in the cartridge aredisposed about a central axis.

[0066] It will be realised that the detector may be positioned relativeto well and light source so as to detect transmitted, reflected,scattered or emitted light.

[0067] Where the detector is a digital camera (or a scanning laser), itmay also be used for assay identification. Thus a bar-code or similarmachine readable code may be placed on the assay cartridge and, readingthis the computer running the apparatus can identify the nature of theassay and hence the assay steps necessary to effect. The assay user cansimilarly apply a bar-code or machine readable code to the assaycartridge to identify the patient so that the apparatus may generate areport identifying patient and assay or may generate an entry in or forthe patient's computerized records. Code-reading and result readingsystems of this nature are discussed for example in WO 98/32004.

[0068] As mentioned above, cartridges in which the pipette iscapillary-tipped rather than membrane tipped may conveniently be usedfor assaying for coagulation time in blood or plasma (preferably blood).The pipette conveniently comprises in sequence a capillary tip, achamber and a second capillary, which may be non-linear, e.g. sinuous,if desired. Opening the cartridge and dipping the capillary tip in ablood sample causes it to fill up to the junction with the chamber, i.e.to take up a predetermined sample volume. The cartridge may then beclosed and placed in the assay device. The second capillary or one ofthe wells in the cartridge is coated with a clot-promoting agent (e.g.tissue factor) and the liquid sample may be contacted with this byapplication of sub-ambient or above ambient pressure respectively to theopen end of the pipette. In the first case, the pressure causes thesample to be drawn through the chamber into the second capillary and sointo contact with the clot-promoting agent. In the second case, thepressure applied expels the sample into the coated well. If desired, inthis second case, the sample and clot-promoting agent may be mixed bybeing drawn back into the pipette and expelled again one or more times.Thereafter the sample is drawn through the capillary tip and chamberinto the second capillary. In both cases, the motion of the sample inthe second capillary under applied pressure is monitored by the detectoruntil clotting has proceeded to the extent that motion is no longerdetectable. This may require the sample to be shuttled back and forth inthe second capillary by alternate application of below and above ambientpressures.

[0069] It will be appreciated therefore that the same capillary can beused for collecting the sample (e.g. blood), and mixing it with one ormore reagents (e.g. by pumping it into and out of a well in thecartridge).

[0070] In any event, for clot time measurements it is important for thesample temperature to be controlled and thus it is desirable that thedevice, e.g. in the cartridge holder, be provided with temperaturecontrol, e.g. a thermostated hot-plate, a hot air source, etc.

[0071] In an alternative embodiment, clotting time in blood or plasmamay be determined by depositing the sample into a well containing aneffervescent agent and monitoring the rate of rise of the bubblesgenerated using a digital camera.

[0072] Where a capillary tipped pipette is used, it may be desirable forthis to be provided separate from the cartridge, formed to bepositionable in a well and couplable to the pressure applicator.

[0073] Such capillary-tipped pipettes and their use in conjunction withassay cartridges form further aspects of the invention.

[0074] Thus viewed from a further aspect the invention provides an assayapparatus comprising:

[0075] i) an assay cartridge comprising at least one, and preferably atleast two, wells and a pipette positionable in at least one, andpreferably at least two, of said wells, said pipette having a capillarytip;

[0076] ii) a holder arranged to receive said cartridge;

[0077] iii) drive means operable to position said pipette in selectedwells of said cartridge;

[0078] iv) a gas pressure applicator couplable to said pipette wherebyto cause liquid flow through said membrane; and

[0079] v) a radiation detector operable to detect radiation from a wellof said cartridge or from said pipette. Viewed from a still furtheraspect the invention also provides an assay cartridge comprising atleast one, and preferably at least two, wells and a pipette positionablein at least one, and preferably at least two, of said wells, saidpipette having a capillary tip.

[0080] Using the pipettes in the assay cartridges of the invention, itis thus possible to introduce test samples into cartridge wells, to mixreagents or reagents and sample in the wells, to transfer liquids fromone well to another, etc. By pumping liquids in and out of a pipette inone well it is possible to improve homogeneity of mixing and by pumpingliquids back and forth across a reagent-carrying pipette membrane it ispossible to increase the extent of the reaction with the reagent. Byvarying the-rate at which a liquid is pumped across a reagent-carryingpipette membrane it is also possible to vary the extent to which thereagent reacts. Accordingly the pipette and cartridge format gives greatversatility for assay performance.

[0081] Where the assay cartridge includes a capillary tipped pipette,e.g. for conveying blood samples, it is frequently desirable to removeexcess fluid from the outer surface of the capillary. In such cases, itis preferred that one of the wells be provided with an absorbent pipettewiper against which the capillary tip may be drawn so as to cause thewiper to absorb any fluid on the outer surface of the capillary. Thiswiper may for example take the form of an absorbent pad disposed at ornear the upper end of the well, e.g. a U shaped pad, preferably notchedat the base of the U. In such an embodiment, as the capillary iswithdrawn from the well it may be displaced sideways to engage thecapillary tip with the notch. Since such displacement may occur beforethe membrane-tipped pipette is fully withdrawn from the well in which itis disposed, it may be necessary to design the wells to prevent themembrane-tipped pipette from being driven into a well side wall. Thusthe well for the membrane tipped pipette may be made wider oralternatively its side wall may be partially removed at the upper end ofthe well.

[0082] Rather than wiping a capillary tip to remove excess sample fromthe outside of the tip, an alternative is to insert the capillary tipinto an absorbent array disposed parallel with the axis of the capillarytip, e.g. absorbent fibres lying parallel to the tip or sheets ofabsorbent material (e.g. paper) with surfaces parallel to the capillarytip axis. Since the open tip of the capillary will not contact theabsorbent material, the contents of the capillary are not removed whilethe outside of the capillary is cleared of excess fluid. This isparticularly important with blood samples. Thus for example a 1 μLcapillary shows poor precision unless the blood sticking to the outsideof the capillary is removed. On an average a 1 μL capillary carries 0.25μL on the outside. Without removal of blood sticking to the outside a CV(coefficient of variation) of about 7-8% (volume of blood delivered) isfound. With efficient removal of blood carried on the outside the CV isreduced to 1.0-1.5%.

[0083] Where capillary wiping takes place as part of assay performance,the time delay before wiping occurs may lead to drying of the blood onthe outside of the capillary. When this happens the blood will not allbe absorbed and may be solubilized during a subsequent dilution step. Ifthe user waits one minute from taking the blood into the capillary tostarting the instrument, the wiping off is somewhat inefficient. Waitingthree minutes means no absorption of blood at all.

[0084] It is therefore greatly preferable if capillary wiping takesplace immediately after blood sample uptake by the capillary. This canbe achieved by disposing in a capillary-receiving well of the cartridgean absorbent array as described above, e.g. a strip of paper folded intoa V-shape with the open end of the V receiving the capillary tip. Thepaper may be positioned and kept stable in the well either by using theforces of the paper pushing outwards against the well walls or ifnecessary by mounting the paper in a supporting frame. When the userintroduces the capillary holder into the cartridge, the capillary willpush the two upper arms apart and the capillary will slide down incontact with the paper on two sides opposite to each other. Thisconstruction with the paper parallel to the capillary ensures that noblood can be absorbed from the interior of the capillary and in additionthe capillary will never hit the bottom part of the folded paper. Usinga 1 μL capillary and whole blood, a CV (blood volume) of 0.75% wasachieved with this construction.

[0085] In a further preferred embodiment, the assay cartridge isprovided to the user with a capillary-tipped pipette to be used forsample taking either loose or detachably mounted in the cartridge, e.g.in an end well of a linear well array. In this embodiment, detachablymounted on the capillary tip, i.e. the distal end of the pipette, is asleeve which closely engages and is preferably flush with the open endof the capillary-tip. On sample uptake by the capillary, any excessexternal liquid accordingly sticks to the outside of the sleeve ratherthan to the outside of the capillary proper. The sleeve is preferablyprovided, e.g. on its external surface, with means to engage with theinner or upper surface of a well in the cartridge (e.g. a distortableflange, etc.) so that when the loaded capillary-tipped pipette ispressed into that well the capillary-tipped pipette can then be removedfrom the well (e.g. on commencement of automated assay performance)leaving the sleeve and the excess external liquid behind in the well.Experiments have shown that, in transferring a 1 μL blood sample usingsuch a sleeve protected capillary, CV (blood volume) as low as thoseachievable with the folded paper wiper described in the previousparagraph can be achieved.

[0086] For certain assays, it may be desirable to carry out a separationof the sample, e.g. to generate a plasma sample from an original bloodsample. In such cases it may be desirable to place a filter in one ofthe wells. This may be removable or alternatively may form part of anintegral pipette extension seated in the well. Such a pipette extensionmay for example comprise a cylinder open at its upper end where it isshaped for engagement with a pipette mounted on the cartridge cap, andpacked at its lower end with glass fibre. In one such embodiment, thesample may be taken up into a capillary tipped pipette mounted on thecartridge cap when cap and base are separated or into a capillary tippedpipette mountable in the cartridge cap. Then, with the cap and baseengaged, the sample may be expelled under air pressure into the cylinderof the pipette extension; the filtrate will pass into the base of thewell. A second cap-mounted capillary tipped pipette can then be used todraw up the filtrate after the pipette and pipette extension have beenwithdrawn from the well. In this way, starting from a blood sample, anundiluted plasma sample may be produced.

[0087] As well as pipette extensions, capillary wipers, etc., otheritems may be disposed within the wells of the cartridge. Thus forexample the well for receiving a sampling capillary may contain afurther fixed or removeable well containing a dried reagent so that thesample and this reagent may be mixed at the onset of assay performance.

[0088] The apparatus, device and cartridges of the invention are for usein assay methods. Such methods, using the apparatus, device orcartridges of the invention form a further aspects of the invention.While the invention is particularly suited for medical diagnosticassays, it can also be used for other assays, e.g. environmental,nutritional, etc., including assays of samples from manufacturingprocesses. It is particularly suitable for such uses as the cartridgesand devices can be produced sufficiently small as to be fully portable,e.g. with the maximum dimension of the device (excluding any connectorsto external equipment or power sources) being no more than 30 cm, morepreferably no more than 20 cm.

[0089] The use of membrane-tipped pipettes in assays is also novel andforms a further aspect of the invention. Viewed from this aspect theinvention provides an assay method wherein a liquid is transferred froma container into a pipette, characterised in that the end of saidpipette through which liquid enters is sealed by a liquid permeablemembrane.

[0090] Viewed from another aspect the invention also provides the use ofthe apparatus of the invention to assay for an analyte in a biologicalsample or for a property of a biological sample, e.g. to assay forclotting time in a blood or blood-derived sample or to assay for aprotein analyte in a body fluid or body fluid-derived sample.

[0091] Documents referred to herein are incorporated herein byreference.

[0092] Examples of apparatus and methods according to the invention willnow be illustrated further with reference to the following non-limitingExamples and the accompanying drawings, in which:—

[0093]FIG. 1 is a schematic cross-section through a cartridge accordingto the invention;

[0094]FIG. 2 is a schematic partial cross-section through a cartridgeaccording to the invention;

[0095]FIG. 3 is a schematic partial cross-section through a cartridgeaccording to the invention;

[0096]FIG. 4 is a schematic drawing of apparatus according to theinvention;

[0097]FIG. 5 is a schematic cross-section through a cartridge accordingto the invention.

[0098]FIGS. 6 and 7 show dose-response curves for the assays of Examples1 and 2;

[0099]FIG. 8 shows the results of the assay of Example 3;

[0100] FIGS. 9 to 19 are schematic views of further embodiments ofcartridges according to the invention in which the wells are arranged ina linear array;

[0101]FIG. 20 is a schematic view showing how a movable magnet may beused to separate magnetic polymer beads from a sample in a well of acartridge according to the invention;

[0102]FIG. 21 is a schematic view showing how a paper strip may be usedto wipe excess liquid off the outside of a capillary-tipped pipette in acartridge according to the invention;

[0103]FIG. 22 is a schematic view showing how a membrane sealed wastereservoir may form part of a pipette in a cartridge according to theinvention; and

[0104]FIG. 23 is a schematic cross-sectional side view of acapillary-tipped pipette for use in an assay cartridge according to theinvention.

[0105] Referring to FIG. 1, there is shown a transparent plasticcylindrical cartridge base 1 containing cylindrical wells 2 (only two ofwhich are shown) disposed in a circular array about cartridge axis 3.Above cartridge base 1 is disposed cartridge cover 5. The mouths of eachwell are sealed by stoppers 4 attached to the cover 5. Cover 5 alsoholds pipette 6, presenting a pressure applicator attachment extension 7to the outside of the cover and with membrane 8—tipped pipette enddisposed in a well 2 of the base 1. A sample introduction port 9 is alsopresent in the cover 5. Port 9 and pipette 6 are kept in registry withwells 2 by mating projections and recesses 10, 11, 12, 13. Similarmating projections and/or recesses 14 (here shown as recesses) areprovided in base 1 and cover 5 to allow base and cover to engage withcartridge holder and drive means (not shown) of the assay apparatus.Base and cover are provided with flanges 15 to engage with the separator(not shown) which pushes base and cover seals 16 apart before assayperformance begins. The nature of the assay for which the cartridge isintended is identified by a bar-code label 17 on the side of the base.The pipette and sample application port are shown sealed by removablestrip seals 16. These are removed before the cartridge is used.

[0106] In FIG. 2, the cartridge of FIG. 1 is shown in a differentorientation for assay result reading at the end of assay performance. Inthis orientation, the wells 18 and 19 shown are different from the wells2 in FIG. 1. Well 18 is a “reading well” having a plastic prism 20placed at its base and part of the light path from membrane to detectoris shown as a dotted line 21. Pipette 7 is shown as containing usedreagent 22. Light source 44 is shown in place inside axial channel 45 inthe cartridge base.

[0107] In FIG. 3 is shown a different embodiment of the cartridge ofFIG. 2 in which the bottom of reading well 18 is stepped and the basebelow reading well 18 is inclined whereby together to form a Fresnelprism 29. Light source 46 is arranged to illuminate the membrane. Inthis embodiment, the pipette 7 is also shown with a relatively largevolume chamber 47. This facilitates retention of the liquids used in theassay in the pipette.

[0108] In FIG. 4 the components of the apparatus of the invention areshown schematically. Cartridge 23 (with base 1, cover 5 and pipette 6)is held by holder 24 and moved by drive means 25. Pipette 6 is connectedvia conduits 26 to piston pumps 27 driven by motor 28. A detector, adigital camera 32, is arranged to detect light from the reading well ofcartridge 23 when the assay is completed and light sources 44 and 46with power supply 34 are arranged to illuminate the reading well.

[0109] Drive means 25, motor 28, camera 32 and power source 34 areoperated by computer 35 which provides an output on monitor/printout 36or to remote computer 37 (e.g. via an infra-red wireless connection).Camera 32, light sources 44 and 46, holder 24 and cartridge 23 arewithin a light-tight chamber 38 provided with a cartridge loading andunloading port 39.

[0110]FIG. 5 shows a cross-section through an alternative,capillary-tipped pipette usable in the cartridges of the invention.

[0111] Open pipette-end 39 is adapted to be attached to the pressureapplicator. The other pipette end is provided with a capillary tip 40which communicates to chamber 41 and thence via a further sinuouscapillary 42 to open-end 39. Part 43 of the base of well 2 is coatedwith a coagulation promoting agent, e.g. tissue factor. Dipping thecapillary tip 40 into blood or plasma causes a fixed volume sample to bedrawn in by capillary action. Withdrawing the pipette from the sampleand then either expelling the content into the clot-promoting agentcoated well and then sucking the sample back into the capillary orsucking the sample past the tissue factor in the capillary, hastensonset of clotting and the digital camera can be used to determine thetime at which sample flow along capillary 42 effectively ceases, i.e.the clotting time.

[0112] FIGS. 9 to 19 showed alternative arrangements for an assaycartridge in which the wells are arranged in a linear way.

[0113]FIG. 9 shows a detached capillary tipped pipette 50 which may bedipped into a liquid to take up a sample. The loaded pipette may then beslotted into aperture 51 in cartridge cap 52 so disposing the capillarytip in an end well in cartridge base 53. The open upper end of pipette50 is provided with notches 54 so that if the operator engages thepipette with the cartridge cap and base by pressing on the top of thepipette this does not raise the pressure in the pipette and so expelsome or all of the sample prematurely. FIG. 10 shows the cartridge ofFIG. 9 assembled following insertion of the sampling pipette, i.e. atthe stage when the cartridge is ready to be placed in the apparatus ofthe invention.

[0114] During performance of the assay, cartridge cap and base will beseparated by disengagement of latch mechanism 84. The separatedcartridge is shown in FIG. 11. Cartridge cap 52 is shown carryingcapillary tipped pipette 50 and membrane tipped pipette 55. Membranetipped pipette 55 is rectangular in cross-section and has an angled tip56. For clarity, the membrane covering the open lower end of pipette 55is not shown. Cartridge base 53 is shown with six wells 57-62, allgenerally rectangular in cross section. To allow for capillary tipwiping, a portion of the upper section of the wall between wells 57 and58 is absent. As shown in FIG. 12, the bases 63 of wells 59 to 62 areangled so as to be parallel to the tip 56 of the membrane-tippedpipette. Wells 59 to 62 are foil-sealed at their upper ends. The foilseals are pierced during assay performance by piercers 64 initiallymounted in the cartridge cap (see FIG. 13). The individual piercers areconnected together in a strip 65 shown in FIG. 14. Each piercer, whichmay be metal but preferably is plastic, is a hollow rectangularcross-section cylinder with a blade edge 66 on the lower rim and flanges67 on the upper rim which cause the piercer to be retained by thecartridge base once it has been forced into engagement with the base (asshown in FIG. 15). The internal cross section of the piercers is shapedto act as a guide for the pipettes.

[0115]FIG. 16 shows the cartridge cap and base being separated with asideways displacement to bring the capillary tip of pipette 50 intocontact with an absorbent wiper 68 disposed at the top of well 57. Asshown, membrane-tipped pipette 55 is partly displaced from well 58 intowell 57.

[0116]FIGS. 17 and 18 are exploded views of cartridge cap and baseassemblies with pipette extensions 69 and 70 which in use would bedisposed in the well (57) into which the sampling pipette 50 isinitially introduced. In the case of FIG. 18, the pipette extension 70serves to transform the sampling pipette into a membrane tipped pipette,e.g. to allow a sample to be filtered.

[0117]FIG. 19 shows the lower ends of three wells arranged forperformance of blood clotting assays having in FIGS. 19a and 19 b asteel ball 72 movable along the base of the well and in FIG. 19c apolymer ball 73 which will float on the sample surface while it is stillfluid.

[0118] After assay performance using the cartridges of FIGS. 9 to 19, anabsorbent strip is preferably inserted into aperture 71 in the cartridgecap so as to prevent seepage of any fluid remaining in wells 58 to 62.Alternatively, the aperture may be sealed with an elongate “piston”which is used to press the piercers through the foil seals of wells 58to 62.

[0119] In FIG. 20, is shown a well 75 in a cartridge according to theinvention. This well contains a liquid 76 containing magnetic polymerbeads. To separate the beads from the liquid during assay performance(e.g. as in Example 12 below), a magnet 77 is moved from a position (A)in which it is remote from the well to a position (B) in which itcontacts the well wall. A membrane-tipped pipette can then be insertedinto the well and used to withdraw the liquid leaving behind themagnetic beads.

[0120] In FIG. 21, is shown schematically a cartridge 78 according tothe invention with a linear array of wells 79-84, an end one 79 of whichis arranged to receive a sampling capillary the tip 85 of which isshown. Within well 79 is disposed a V-shaped fold of absorbent paper 86such that insertion of capillary tip 85 into well 79 causes the sides ofthe capillary to be wiped.

[0121] In FIG. 22, is shown partially and schematically a cartridge 87according to the invention having capillary-tipped and membrane-tippedpipettes 88 and 89 in cartridge cap 90. The membrane-tipped pipette 89has towards its proximal end a liquid waste reservoir 91 and when inplace within cartridge cap 90 the reservoir is closed by a self-sealingrubber gasket 92. Where pressure is to be applied to the proximal end ofthe membrane-tipped pipette 89 this is done by piercing the gasket 92with a hollow needle 93 attached to a pressure applicator (not shown).

[0122] In FIG. 23 is shown a capillary-tipped pipette 94 which isprovided as part of an assay cartridge according to the invention. Asprovided to the user, pipette 94 is loosely positioned in one well, e.g.as pipette 50 in well 57 in the embodiment of FIG. 11. The distal end 95of pipette 94 is provided with a sleeve 96 which grips the pipette endand closely surrounds and is flush with the very tip of the capillary.The upper rim of sleeve 96 is provided with a distortable flange 97which can be forced past a matching flange in the well so as to lock thesleeve into the well. In use, the capillary-tipped pipette is removedfrom the cartridge with sleeve 96 attached, dipped into a liquid sampleto take liquid into the capillary tip, and replaced in the well andpressed to lock the sleeve into the well. The cartridge may then beloaded into the assay device and in assay operation separation ofcartridge cap and base serves to disengage the sleeve from thecapillary.

EXAMPLE 1 Assay for C-Reactive Protein In Serum

[0123] 1 μl samples of human blood, spiked with purified C-reactiveprotein (CRP) to concentrations ranging from 0 to 160 mg/l are placed ina 9 mm internal diameter, round-bottomed well (in an assay cartridgeequivalent to the cartridge of FIG. 1) containing 200 μL of an aqueousdilution liquid (30 mM borate buffer, pH 8.0 containing 0.01% w/v sodiumcitrate, 0.02% w/v NaN₃ and deoxycholate).

[0124] The membrane-tipped pipette, having an external diameter of 7.2mm, is lowered into the sample-containing well, and below ambientpressure is applied to the open end of the pipette causing the wellcontents to flow through the membrane into the pipette. In this Example,the pipette membrane is a nitrocellulose sheet having immobilizedthereon a monoclonal anti-CRP antibody (prepared by conventionaltechniques).

[0125] The pipette is then removed from the well and lowered into asecond well of the same configuration containing 200 μL of an aqueousdispersion of gold microbeads (average diameter 4.5 nm, concentration(optical density at 540 nm) of about 3, corresponding to an antibodyconcentration of about 50 μg/mL in 50 mM borate buffer pH 8.05,containing 20 mM NaCl, 0.05% w/v NaN₃ and 0.1% w/v BSA) conjugated inconventional fashion to a monoclonal anti-CRP antibody. Below ambientpressure is again applied to the open end of the pipette causing theliquid in the well to pass into the pipette so saturating the membranewith the gold conjugate.

[0126] The pipette is then removed from the second well and lowered intoa third well, again of the same configuration, containing 200 μL of theaqueous dilution liquid (supra) Below ambient pressure is applied to theopen end of the pipette to draw the washing reagent into the pipette; inthis way, unbound gold conjugate is removed from the membrane.

[0127] The pipette is then removed from the third well and placed into afourth, 9 mm internal diameter, flat-bottomed, empty well. For thisassay, this fourth well is the reading well. The pipette membrane isilluminated (e.g. with green light from a LED) through the transparentwell-containing base of the assay cartridge and light of 540 nmreflected by the membrane is detected using a detector (e.g. a digitalcamera or a photodiode).

[0128]FIG. 6 of the accompanying drawings shows the linear dose-responsefor this assay using a green LED.

[0129] Performance of the assay requires about 40 seconds from serumaddition to reflectance determination.

EXAMPLE 2 Assay for Human Serum Albumin in Urine

[0130] Human urine is depleted of human serum albumin (HSA) byultrafiltration and then spiked with purified HSA to concentrationsbetween 0 and 200 mg/L.

[0131] A 10 μL sample of the urine is transferred in a capillary into a9 mm internal diameter, round-bottomed well (in an assay cartridgeequivalent to the cartridge of FIG. 1) containing 200 μL of aqueoussodium phosphate buffer, pH 5.6 containing 4.0% v/v propan-1-ol, 0.05%w/v NaN₃, 0.003% w/v Tropeolin-O and 0.5% w/v BSA. The urine is mixedwith the dilution buffer by being pumped in and out of the capillarythree times. The capillary is removed and the membrane-tipped pipette islowered into the well. In this assay the membrane is a nitrocellulosesheet having immobilized thereon a monoclonal anti-HSA antibody. Thediluted sample is drawn into the pipette as in Example 1.

[0132] The pipette is then removed from the well and lowered into asecond well having the same configuration but containing 200 μL of adispersion of gold microbead-antibody conjugate (as in Example 1 butwith an anti-HSA rather than an anti-CRP antibody, 50 mM borate bufferpH 7.8, 0.05% w/v NaN₃, and 0.2% w/v BSA). The well contents are drawninto the pipette as in Example 1, and as in Example 1 the pipette isthen transferred to a third (washing) and fourth (reading) well. In thisassay the washing reagent is PBS, pH 7.4.

[0133]FIG. 7 of the accompanying drawings shows the dose-response curvefor this assay.

EXAMPLE 3 Assay for Glycated Hemoglobin in Blood

[0134] 1 μL of whole blood is taken from a blood sample using acapillary mounted on the tip of an inverted conical container, volumeabout 500 μL, i.e. a funnel-shaped device, to the upper end of which isattached a pressure applicator.

[0135] The capillary is lowered into a 9 mm internal diameter,round-bottomed well in an assay cartridge (as described for the previousExamples) containing 200 μL of an aqueous boronic acid conjugatesolution.

[0136] The conjugate solution comprises 0.25 mM xylene-cyanole boronicacid conjugate (Example 18 of U.S. Pat. No. 5,631,364), 0.07% w/v TritonX-100, 9 mM zinc chloride, and 100 mM HEPES buffer, pH 8.15.

[0137] The blood sample is pumped into the well and mixed with theboronic acid conjugate solution by pumping the solution into and out ofthe conical container three times. The capillary is removed and the wellcontents are allowed to incubate for two minutes. This permits thedetergent to lyse the blood cells, the zinc to precipitate thehemoglobin and the boronic acid conjugate to bind to glycatedhemoglobin.

[0138] The membrane-tipped pipette is then lowered into the well andbelow ambient pressure is applied causing the liquid in the well to passinto the pipette and the hemoglobin to become trapped on the membrane.In this assay the membrane is a porous filter having a 1 μm pore size.

[0139] The pipette is removed from the well and placed in a second wellof the same configuration containing 200 μL of an aqueous washingreagent (50 mM morpholine buffer, pH 9.5, containing 200 mM NaCl, 0.5%w/v Triton X-100, 0.1% w/v glycerol and 0.05% w/v NaN₃. Below ambientpressure is applied to the pipette drawing the washing reagent andunbound boronic acid conjugate into the pipette.

[0140] The pipette is then removed and lowered into a 9 mm internaldiameter, flat bottomed, empty reading well in the cartridge forreflectometric measurement of the hemoglobin trapped on the pipettemembrane. Total hemoglobin is measured using blue light at 460 nm andglycated hemoglobin using red light at 620 nm (e.g. using red and blueLREDs). The proportion of glycated hemoglobin relative to totalhemoglobin (sometimes referred to as %Hb1Ac) is determined by the ratioof the measured reflectancies, calibrated against samples with known%Hb1Ac.

[0141]FIG. 8 of the accompanying drawings shows the results for theassay of this Example for 6 blood samples analysed for %Hb1Ac 24 hoursearlier using HPLC (Variant, BioRad).

EXAMPLE 4 Liquid Collection Efficiency for Membrane-Tipped Pipettes

[0142] The efficiency of liquid collection from different wellconfigurations was tested for a planar nitrocellulose membrane-tippedpipette as described in Example 1 in comparison with a standard conical,open-tipped pipette. In each case 200 μL of liquid was to be withdrawnfrom a flat or round bottomed 9 mm internal diameter well in a soft orhard plastic base (LDPE and polystyrene respectively). The results areset out in Table 1 below. TABLE 1 % Liquid collected Open-tippedMembrane-tipped Well pipette pipette Soft, round 98.9 99.8 Hard, round99.5 99.7 Hard, flat 84.0 99.5

EXAMPLE 5 Assay for Coagulation Time for Blood

[0143] The pipette of FIG. 5 is used to collect an approximately 2 μLsample of blood. The cartridge is then reassembled and pressure isapplied to the pipette to expel the blood sample into a cartridge well,the base of which is coated with a coagulation promoting agent (e.g.tissue factor). Below ambient pressure is then applied to draw thesample back into the pipette, past the chamber into the sinuouscapillary. The sample is then shuttled back and forth in the sinuouscapillary under the application of above ambient and sub-ambientpressures and, using the digital camera, the time between the bloodsample contacting the coagulation promoting agent and effectivecessation of blood sample movement is determined. This may typically beabout 40 seconds.

EXAMPLE 6 Assay for Coagulation Time for Whole Blood or Plasma

[0144] An assay cartridge of the type shown in FIG. 11 is used. One ofwells 59 to 62 contains dried tissue factor and calcium chloride orgluconate as well as a steel ball, e.g. 2 mm diameter (see FIG. 19a).

[0145] The apparatus into which the cartridge is to be placed isprovided with a heating element to maintain the cartridge contents atabout 37° C. and with a magnet to shuttle the steel ball along the baseof the well in which is disposed.

[0146] In well 57 is disposed a removable capillary tipped pipettecapable of taking up a preset volume of sample, e.g. 1 to 15 μL,preferably 10 μL, of whole blood, citrated venous blood, plasma orcitrated plasma.

[0147] The sample is taken up by the capillary-tipped pipette which isthen placed in the cartridge which is then placed in the assayapparatus. The sample is then transferred into the steel ball-containingwell and mixed.

[0148] The cartridge is then shuttled relative to the magnet in ahorizontal direction parallel with the tip of the ball containing well.(Either the cartridge as a whole or the magnet may be moved—howeverpreferably the cartridge is moved with the magnet serving initially tokeep the steel ball static.)

[0149] A digital camera is used to monitor the position of the steelball. As the mixture begins to coagulate the ball ceases to be staticrelative to the magnet and this is detected by the camera so allowingthe clotting time (from contact of sample with calcium salt solution) tobe determined.

[0150] In an alternative, less preferred, embodiment, the magnet beneaththe cartridge is omitted and the ball is placed in a well with a slopingbase (e.g. as shown in FIG. 19b). Sharp movement of the cartridge in thedirection of the lower end of the base, e.g. through mechanical shock orby activation of an electromagnet to the side of the well, causes theball to move up the sloping base and, before clotting occurs, the ballreturns to the lower end of the base under the action of gravity.

EXAMPLE 7 Assay for Coagulation Time for Whole Blood or Plasma

[0151] An assay cartridge as in Example 6 is used with a low densitypolymer ball (e.g. a polystyrene ball 3-5 mm in diameter) in place ofthe steel ball. This ball is preferably in a flat or concave-bottomed,circular cross section well (see FIG. 19c).

[0152] A sample is taken and mixed as in Example 6 and then placed inthe ball-containing well where the ball will float on the samplesurface. The ball is then repeatedly urged under the sample surface andallowed to float back up to the surface. As the sample coagulates theball will return to the surface more slowly and then not at all.

[0153] The ball may be urged under the surface by pressure from the tipof the pipette or alternatively a magnetically movable ball may be usedand a magnetic field may be switched on and off to draw the ball downand release it respectively. Such magnetically responsive balls may beprepared for example by depositing superparamagnetic crystals in thepolymer ball (e.g. as in the magnetic beads sold by Dynal Biotech, Oslo,Norway).

EXAMPLE 8 Assay of Clotting Time for Plasma

[0154] An assay cartridge similar to that shown in FIG. 11 is used. Asin Example 6, one of wells 59 to 62 contains a citrate buffer, anothercontains fibrinogen and coagulation factor V and a third a calcium saltsolution. Well 57 contains a capillary-tipped pipette and well 58contains a filter extension as shown in FIG. 18.

[0155] A sample is taken up in the capillary tipped pipette which isthen placed in well 57 and the cartridge is placed in the assayapparatus and there warmed to 37° C. The sample is then transferred tothe buffer-containing well and mixed. The whole or a preset proportionof the mixture is then transferred into the filter pipette extension andcell-free diluted plasma is pumped into the base of the well. Apredetermined volume of cell-free plasma is then transferred into thefibrinogen containing well using a further capillary tipped pipette andthis further pipette is also used to transfer a predetermined volume ofthe calcium salt solution to the fibrinogen/plasma containing well toinitiate the clotting-reaction. The well is illuminated and a digitalcamera is used to record the turbidity of the mixture in the well. Thetime from calcium addition to increase of turbidity to a pre-definedvalue is taken as the clotting time.

EXAMPLE 9 Assay for Clotting in Whole Blood or Plasma

[0156] An assay cartridge similar to that shown in FIG. 11 and describedin Example 8 is used. As in Example 8, one of wells 59 to 62 containscitrate buffer and another a calcium salt solution, however theball-containing well is omitted and in place of coagulation factor V andfibrinogen the “reagent” well contains a dried thrombin-specificchromogenic substance (e.g. Nycotest Chrom (described in Janson et al.Thrombostasis and Haemostasis 62: 530 (poster 1677) (1989) and Jonker etal. Research in Clinic and Laboratory 20: 45-57 (1990)) or one of thechromogenic substances discussed in DE-A-3113350, DE-A-3413311,DE-A-3311287, U.S. Pat. No. 4,458,015 or U.S. Pat. No. 4,784,944).

[0157] The sample is taken and mixed analogously to the procedure inExample 7. The coagulation process results in thrombin formation andthus the release of a dye from the chromogenic substance (e.g. yellowpara-nitroaniline from Nycotest Chrom).

[0158] The change in colour of the sample is followed using the digitalcamera and the clotting time is taken as the time from calcium additionto a predetermined colour change.

EXAMPLE 10 Assay for C-Reactive Protein (CRP) in Whole Blood UsingEnzyme Conjugate (ELISA)

[0159] Using the capillary-tipped pipette of the cartridge; 1 μL wholeblood is added to a well (e.g. well 59) of a cartridge similar to thatshown in FIG. 11 and containing 200 μL of a dilution and lysing liquid(30 mM borate buffer pH 8.0 containing 0.01% w/v sodium citrate, 0.02%w/v NaN₃ and dexoycholate). The wells of the cartridge have arectangular cross section with inner dimensions 6.0 by 6.5 mm. Theplanar bottom of the well is angled 30 degrees to the length axis of thewell.

[0160] The rectangular membrane-tipped pipette (which has outerdimensions 3.7 by 4.2 mm and is equipped with an anti CRPantibody-coated nitrocellulose membrane mounted 30 degrees to the lengthaxis of the membrane tube) is lowered into the well and the lysed bloodcell solution is absorbed through the membrane by applying below ambientpressure to the interior of the membrane-tipped pipette. When all liquidis absorbed, an above ambient pressure is applied to force the liquid asecond time through the membrane and back into the well. Passing the CRPsolution twice through the membrane increases further the captureefficiency of CRP.

[0161] Subsequently the membrane-tipped pipette is moved to a similarwell (e.g. well 60) in the cartridge which contains a solution ofalkaline phosphatase (ALP) conjugated to an anti CRP antibody(approximately 40 μg/ml ALP and 40 μg/ml antibody in 50 mM borate bufferpH 8.0 containing 0.02% w/v NaN₃ and 0.5% w/v BSA. The conjugatesolution is absorbed through the membrane and pumped back into the wellby applying a sequence of below and above ambient pressure inside themembrane-tipped pipette as described above for the antigen capture.

[0162] In the next step, the membrane-tipped pipette is moved to afurther well (e.g. well 61) in the cartridge which contains 200 μL ofwashing solution (50 mM borate buffer pH 8.0 containing 0.01% w/v NaN₃,0.5% w/v BSA and deoxycholate) which is absorbed and subsequently pumpedback into the well. This washing step is repeated twice by moving themembrane-tipped pipette to two additional wells (not shown in FIG. 11but equivalent to well 61) which also contain the washing solution. Thetotal of three washing cycles ensures an efficient removal of unboundconjugate.

[0163] Finally the membrane-tipped pipette is moved into a still furtherwell (e.g. well 62) in the cartridge which contains 300 μL of a solutionof the alkaline phosphatase substrate para nitrophenyl phosphate (1.0mg/ml pNPP in 1.0 M diethanolamine buffer pH 9.6 containing 0.5 mM MgCl₂and 0.025% w/v NaN₃). The yellow enzyme product para-nitrophenol isdeveloped by pumping the substrate solution in and out of themembrane-tipped pipette over a period of two minutes. The incubation isterminated by pumping all liquid back into the well and raising themembrane-tipped pipette out of the substrate solution. Using 300 μL ofsubstrate solution the filling height is about 3 mm above the top of theangled part of the well, thus allowing the colour to be measured throughparallel walls of the well.

[0164] With the membrane-tipped pipette raised, the absorbance ismeasured using a blue LED as a light source and a digital camera formeasurement of transmitted light.

EXAMPLE 11 Assay for C-Reactive Protein (CRP) in Whole Blood Using LightScatter Measurement of Aggregated Latex Beads

[0165] Using the capillary-tipped pipette of the cartridge, 2 μL wholeblood is added to a well (e.g. well 62) of a cartridge similar to thatshown in FIG. 11 and containing 120 nm Latex beads (0.2% w/v) suspendedin 300 μL 50 mM borate buffer pH 8.0 containing 0.01% w/v sodiumcitrate, 0.02% w/v NaN₃ and deoxycholate. The beads are coated by simpleadsorption with anti CRP antibodies. The well has a rectangular crosssection and is at the end of the cartridge to facilitate the measurementof light scatter. Light is directed onto one side wall of the well.After an initial phase of cell lysis which takes about 10 seconds, theincrease of light scatter is measured at an angle of 90 degrees to theincident light. The increase of light scatter due to the CRP-mediatedaggregation of the Latex beads is measured by the digital camera at awavelength of 425 nm.

EXAMPLE 12 Assay for Albumin in Urine Using Magnetic Beads ColouredLatex Beads and Relectometry

[0166] Using the capillary-tipped pipette of the cartridge, 2 μL urineis added to a well (e.g. well 62) of a cartridge similar to that shownin FIG. 11 and containing 1000 nm magnetic polymer beads (0.2% w/v) and1000 nm blue Latex beads (0.2 L w/v) in 200 μL 30 mM sodium phosphatebuffer pH 5.7 containing 0.5% w/v BSA and 0.05% w/v NaN₃. The magneticbeads (e.g. of the type available from Dynal Biotech, Oslo, Norway) arecoated with an antibody reacting with an epitope on the albumin moleculedifferent from the epitope recognized by the antibody coated onto theLatex beads.

[0167] After incubation for 60 sec, a Neodymium magnet (10×7×2 mm) ismoved from its resting position (20 mm from the nearest wall of thewell) towards the well to bring the magnet in direct contact with theside wall of the well. The magnet makes contact with the wall oppositeto the angled one and covers the liquid filled part of the well (200μL). The well and the positioning of the magnet are shown schematicallyin FIG. 20. In the resting position the magnetic field working on themagnetic beads is too weak to move the beads. When in contact with thewell, the distance from the magnet to the nearest and remotest innerwall of the well is 0.8 mm and 6.3 mm respectively. At this distance thebeads are quantitatively collected on the wall after 30 sec. In thepresence of analyte, blue Latex is linked to magnetic particles and thereacted fraction of the Latex beads will be collected on the wall whileunreacted Latex particles will remain suspended.

[0168] With the magnet in contact position, the capillary-tipped pipetteis used to suck up the liquid containing the unreacted Latex particles.The magnet is then moved away from the well to its resting position.

[0169] The capillary-tipped pipette tube is then moved into an emptywell (e.g. well 61) and the liquid is delivered to this well by applyingabove ambient pressure to the interior of the pipette.

[0170] The capillary-tipped pipette is then moved to a further well(e.g. well 60) which contains 500 μL of washing solution (PBS, pH 7.4)and 200 μL is taken up. The capillary-tipped pipette is then moved backto the well containing the magnetic beads and the beads are suspended bypumping the washing solution in and out of the well five times. Themagnet is moved into the contact position and the magnetic beads areallowed to be collected on the wall of the well. After 30 sec thewashing solution is taken back into the capillary-tipped pipette. Themagnet is subsequently moved back to its resting position.

[0171] The capillary-tipped pipette is in-the next step moved to thewell containing the first supernatant (well 61) and pumped into thiswell.

[0172] The capillary-tipped pipette is subsequently moved to the wellcontaining the washing solution (well 60) and 200 μL are taken up.

[0173] The capillary-tipped pipette is moved to the well containing themagnetic beads (well 62) and the beads are resuspended by pumping thewashing solution in and out 5 times.

[0174] A membrane-tipped pipette equipped with a 0.45 μm microporousmembrane is moved to the well containing the suspended magnetic beads(well 62) and the beads are collected onto the membrane by suction.

[0175] The membrane-tipped pipette is raised out of well 62 and blueLatex particles and the yellow-brown magnetic beads are quantified byreflectometry using a red LED for the blue Latex beads and a blue LEDfor the magnetic beads. The amount of absorbed red light/amount ofabsorbed blue light is a measure of the fraction of blue Latex in themixture and hence a measure of the amount of albumin present in thesample.

[0176] The same cartridge may also be used for determination ofcreatinine content of urine and hence the albumin:creatinine ratio inthe urine sample. Albumin in urine provides an indicator of kidneyfunction and the albumin:creatinine ratio may be used to correct fordiuresis. Albumin:creatinine measurement is described for example inU.S. Pat. No. 5,385,847.

[0177] In this embodiment a fraction of the urine sample is mixed with adilution reagent and an enzyme or enzyme mixture which reacts withcreatinine to generate a coloured analyte which is detected using adigital camera by measurement of light transmission through a wellcontaining urine, enzymes and dilution reagent.

1. An assay apparatus comprising: i) an assay cartridge (52,53)comprising at least two wells (57-62) and a pipette (55) positionable inat least two of said wells, said pipette having a proximal end and adistal end, said distal end being closed by a liquid permeable membrane;ii) a holder arranged to receive said cartridge; iii) drive meansoperable to position said pipette in selected wells of said cartridge;iv) a gas pressure applicator couplable to said pipette whereby to causeliquid flow through said membrane; and v) a radiation detector operableto detect radiation from a well of said cartridge or from said pipette.2. An assay apparatus comprising: i) an assay cartridge (52,53)comprising at least one well (57-62) and a pipette (50) positionable inat least one said well, said pipette having a capillary tip; ii) aholder arranged to receive said cartridge; iii) drive means operable toposition said pipette in selected wells of said cartridge; iv) a gaspressure applicator couplable to said pipette whereby to cause liquidflow through said membrane; and v) a radiation detector operable todetect radiation from a well of said cartridge or from said pipette. 3.An assay apparatus comprising: i) an assay cartridge (52,53) comprisingat least one well (57-62) and a pipette (50) positionable in at leastone said well, at least one said well having two parallel planar sidewalls joined by a base wall comprising at least one planar face thenormal to the surface whereof is coplanar to and non-perpendicular tonormals to the parallel planar surfaces of said side walls; ii) a holderarranged to receive said cartridge; iii) drive means operable toposition said pipette in selected wells of said cartridge; iv) a gaspressure applicator couplable to said pipette whereby to cause liquidflow through said membrane; and v) a radiation detector operable todetect radiation from a well of said cartridge or from said pipette. 4.Apparatus as claimed in any one of claims 1 to 3 wherein said cartridgecomprises a capillary-tipped pipette (50) and a membrane-tipped pipette(55).
 5. Apparatus as claimed in any one of claims 1 to 4 wherein saidcartridge comprises a pipette (55) the distal end whereof is closed by asloping liquid permeable membrane.
 6. Apparatus as claimed in claim 5wherein said sloping membrane lies in a plane at an angle of 20 to 40°to the axis of the pipette to which it is attached.
 7. Apparatus asclaimed in any one of claims 1 to 6 wherein said cartridge comprises amembrane-tipped pipette the membrane-tipped end whereof is ofrectangular cross section.
 8. Apparatus as claimed in any one of claims1 to 7 wherein said cartridge comprises detachable base and cap members,said wells being disposed in said base member and said cap member beingarranged to carry said pipette.
 9. Apparatus as claimed in claim 8wherein said cap member comprises means to receive a capillary-tippedpipette.
 10. Apparatus as claimed in either one of claims 8 and 9wherein at least-one of said wells is sealed at its upper end by afrangible seal and wherein said cap member is provided with a cutterarranged to pierce said seal.
 11. Apparatus as claimed in any one ofclaims 8 to 10 wherein said base member comprises an absorbent wiperarranged to wipe the outside of a capillary-tipped pipette insertedtherein.
 12. Apparatus as claimed in any one of claims 1 to 11 whereinsaid cartridge comprises a membrane-tipped pipette the proximal endwhereof is closed by a piercable self-sealing membrane.
 13. Apparatus asclaimed in any one of claims 1 to 12 wherein the wells in said cartridgeare arranged in a linear array.
 14. Apparatus as claimed in any one ofclaims 1 to-13 wherein said radiation detector comprises a digitalcamera.
 15. Apparatus as claimed in claim 14 wherein the base wall of atleast one of the wells in said cartridge is planar and non-perpendicularto the contiguous side walls of the well.
 16. Apparatus as claimed inany one of claims 1 to 15 further comprising a light source arranged toilluminate said cartridge.
 17. Apparatus as claimed in any one of claims1 to 16 further comprising a magnet.
 18. Apparatus as claimed in any oneof claims 1 to 17 further comprising a heater arranged to heat saidcartridge.
 19. Apparatus as claimed in any one of claims 1 to 18 furthercomprising a controller arranged to control assay performance by saidapparatus.
 20. Apparatus as claimed in any one of claims 1 to 19 whereinsaid gas pressure applicator comprises a piston disposed within acylindrical housing and a drive motor arranged to drive said piston. 21.An assay cartridge (52,53) comprising at least two wells (57-62) and apipette (55) positionable in at least two of said wells, said pipettehaving a proximal end and a distal end, said distal end being closed bya liquid permeable membrane.
 22. An assay cartridge (52,53) comprisingat least one well (57-62) and a pipette (50) positionable in at leastone said well, said pipette having a capillary tip.
 23. A cartridgeas-claimed in claim 22 wherein said capillary tip (95) is provided witha detachable sleeve (96).
 24. An assay cartridge (52,53) comprising atleast one well (57-62) and a pipette (50) positionable in at least onesaid well, at least one said well having two parallel planar side wallsjoined by a base wall comprising at least one planar face the normal tothe surface whereof is coplanar to and non-perpendicular to normals tothe parallel planar surfaces of said side walls.
 25. An assay cartridgeas claimed in any one of claims 21 to 24.wherein said cartridgecomprises a capillary-tipped pipette (50) and a membrane-tipped pipette(55).
 26. An assay cartridge as claimed in any one of claims 21 to 25wherein said cartridge comprises a pipette (55) the closed end whereofis closed by a sloping liquid permeable membrane.
 27. An assay cartridgeas claimed in claim 26 wherein said membrane-lies in a plane at an angleof 20 to 40° to the axis of the pipette to which it is attached.
 28. Anassay cartridge as claimed in any one of claims 21 to 27 wherein saidcartridge comprises a membrane-tipped pipette the membrane-tipped endwhereof is of rectangular cross section.
 29. An assay cartridge asclaimed in any one of claims 21 to 28 wherein said cartridge comprisesdetachable base and cap members, said wells being disposed in said basemember and said cap member being arranged to carry said pipette.
 30. Anassay cartridge as claimed in claim 29 wherein said cap member comprisesmeans to receive a capillary-tipped pipette.
 31. An assay cartridge asclaimed in either one of claims 28 and 30 wherein at least one of saidwells is sealed at its upper end by a frangible seal and wherein saidcap member is provided with a cutter arranged to pierce said seal. 32.An assay cartridge as claimed in any one of claims 21 to 31 wherein saidbase member comprises an absorbent wiper arranged to wipe the outside ofa capillary-tipped pipette inserted therein.
 33. An assay cartridge asclaimed in any one of claims 21 to. 32 wherein said cartridge comprisesa membrane-tipped pipette the proximal end whereof is closed by apiercable self-sealing membrane.
 34. An assay cartridge as claimed inany one of claims 21 to 33 wherein the wells in said cartridge arearranged in a linear array.
 35. An assay cartridge as claimed in any oneof claims 21 to 34 wherein at least one of said wells contains an assayreagent.
 36. An assay device comprising a) a cartridge holder (24)capable of receiving an assay cartridge (23) according to any one ofclaims 20 to 33; b) drive means (25) operable to position the pipette(6) of a said cartridge in selected wells of said cartridge; c) a gaspressure applicator (27) couplable to the pipette of a said cartridgewhereby to cause liquid flow therethrough the membrane thereof; and d) aradiation detector (32) operable to detect radiation from a well of asaid cartridge or from the pipette thereof.
 37. A device as claimed inclaim 36 wherein said radiation detector comprises a digital camera. 38.A device as claimed in either of claims 36 and 37 further comprising alight source arranged to illuminate said cartridge.
 39. A device asclaimed in any one of claims 36 to 38 further comprising a magnet.
 40. Adevice as claimed in any one of claims 36 to 39 further comprising aheater arranged to heat said cartridge.
 41. A device as claimed in anyone of claims 36 to 40 further comprising a controller arranged tocontrol assay performance by said apparatus.
 42. A device as claimed inany one of claims 36 to 41 wherein said gas pressure applicatorcomprises a piston disposed within a cylindrical housing and a drivemotor arranged to drive said piston.
 43. An assay method wherein aliquid is transferred from a container into a pipette, characterised inthat the end of said pipette through which liquid enters is sealed by aliquid permeable membrane.
 44. A pipette the distal end whereof iscylindrical and tipped by a porous membrane the outer surface whereof isangled away from the plane perpendicular to the cylindrical axis of saiddistal end.
 45. The use of apparatus as claimed in any one of claims 1to 20 to assay for an analyte in a biological sample or for a propertyof a biological sample.
 46. Use as claimed in claim 45 to assay forclotting time in a blood or blood-derived sample.
 47. Use as claimed inclaim 45 to assay for a protein analyte in a body fluid or bodyfluid-derived sample.